Experimental Study Of Repairing Peripheral Nerve Defects With Tetramethylpyrazine And Tissue-engineered Nerve

Part1EFFECT OF TETRAMETHYLPYRAZINE ONNEURAL STEM CELLS WITH GENETICALMODIFICATION OR ISCHEMIA-LIKE INJURYObjective: To explore the protective effects of TMP on neural stemcells(NSCs) genetically modified or cultured in the presence of oxygenglucose deprivation (OGD) condition. To investigate the mechanism oftetramethylpyrazine (TMP) on protecting NSCs and prompting neuralrepair.Methods:(1) The second passage of primary NSCs isolated fromneonatal SD rat was examined and characterized throughimmunohistochemistry at first. The second passage of primary NSCs weregenetically modified (GM) by retrovirus with EGFP gene in vitro. Then,its were sequently cultured for72hours in the presence of TMP(0,50,100,200or300μg/ml) or0.1%DMSO (vehicle control) in normal conditionsin vitro, meanwhile, culturing second passage of primary NSCs as thenormal control. Cell viability and cell death was determined through the3-[4, 5-dimethylthiazol-2-yl]-2,5-dipheny-ltetrazolium bromide (MTT) assay,flow cytometry, and trypanblau dyeing count. The expression of Bax, Bcl-2,and Caspase-3were investigated by real-time PCR and western immunoblotanalysis.(2)The second passage of primary NSCs, after cultured72hours,were exposed to oxygen glucose deprivation(OGD) for6hours in thepresence of TMP (0,50,100,200or300μg/ml) or0.1%DMSO (vehiclecontrol) in vitro. meanwhile, using cells cultured in normal conditions for6hours as the normal control. Cell viability and cell death was determinedthrough the MTT assay, flow cytometry, and trypanblau dyeing count. Theexpression of Bax, Bcl-2, and Caspase-3were investigated by real-time PCRand western immunoblot analysis.Results:(1) After genetically modified, the Cell viability of NSCs invehicle control and GM control significantly decreased, while cell death andapoptosis dramatically increased（all of P＜0.05）. It also induced asignificant decreases in the mRNA and protein expression levels of Bcl-2and an notable increase in the mRNA and protein expression levels of Baxand Caspase-3（all of P＜0.05）. But there was no statistical differencesbetween vehicle control and GM control（all of P＞0.05）. Notably,compared with GM control, TMP treatment reduced cell death, amelioratedcell viability losses, suppressed cells apoptosis in a concentration-dependentmanner. Moreover, the down-regulation of Bcl-2and up-regulation of Baxand Caspase-3were attenuated by TMP in a concentration-dependent manner. But there were no statistical difference among GM control,50μg/ml TMP and100μg/ml TMP group(P＞0.05), likewise, no statisticaldifference were found between200μg/ml TMPand300μg/ml TMPgroup (P＞0.05).(2) After exposed to OGD, the Cell viability of NSCs in vehiclecontrol and GM control significantly decreased, while cell death andapoptosis dramatically increased（all of P＜0.05）. It also induced asignificant decrease in the mRNA and protein expression levels of Bcl-2andan notable increase in the mRNA and protein expression levels of Bax andCaspase-3（all of P＜0.05）. But there was no statistical differentiationbetween vehicle control and OGD control（all of P＞0.05）. Notably,compared with OGD control, TMP treatment reduced cell death,ameliorated cell viability losses, suppressed cells apoptosis in aconcentration-dependent manner. Moreover, the down-regulation of Bcl-2and up-regulation of Bax and Caspase-3were attenuated by TMP in aconcentration-dependent manner. But there were no statistical differenceamong OGD control,50μg/ml TMP and100μg/ml TMP group(P＞0.05),likewise, no statistical difference were found between200μg/ml TMP and300μg/ml TMP group (P＞0.05).Conclusions: TMP treatment can reduced cell death, ameliorated cellviability losses, suppressed cells apoptosis in a concentration-dependentmanner after NSCs genetically modified or exposed to OGD. Moreover, it’srelative with the down-regulation of Bcl-2and up-regulation of Bax and Caspase-3in the mRNA and protein expression levels. It may is one ofmechanism of TMP providing protective effects in peripheral nervoussystem injury and regeneration.PART2THE EFFECT OF TMP ON ACELLULARNERVE ALLOGRAFT TRANSPLANTED WITHCULTURED NERVE STEM CELLS OF RATPurpose: To chemically extract acellular nerve allograft, constructtissue-engineered nerve bridging graft with acellular nerve transplantedwith cultured nerve stem cells, approach the effect of bridging allograft byTMP, provide a ideal bridging allograft for repairing peripheral nervousdefection.Methods:(1)Take24nerve segments from both-side sciatic nerves of12healthy adult male Wistar rats，20nerve segments of them get achemical treatment as following:①Strip overlying connective tissue, washthem3times with sterile distilled water.②After being shaken24hours in3%TritonX-100,wash them3times with sterile distilled water.③Afterbeing shaken24hours in4%sodium deoxycholate and roomtemperature,wash them3times with sterile distilled water.④Repeat steps2-3, till the last acellular nerve were milk white and translucent. Then,observe structure of the acellular nerve tissues in HE staining and scanning electron microscopy.⑤Reserve the acellular nerves in the DMEM/F12medium at4℃.(2)12hydrated acellular nerves segments, after beinginjected10ul(2×106/ml) genetically modified neural stem cell suspensionwith a micro pipette under the microscope,divided into I、II、III groups(5samples in I groups,5samples in II groups,2samples in III groups), I andII groups were cultured1weeks in conventional DMEM/F12medium(Igroup) or adding200ug/ml TMP(II group). Then, observe cells distributionand framed construction through the line fluorescence microscope, HEstaining and immunohistochemical (Nestin, NF-200, GFAP) staining.（3）Take24healthy adult male SD rats equally divide into A、B、C、D4groups,4sorts(24sticks) nerves segments(sciatic nerves reserved in4℃for1week of Wistar rat, I group tissue-engineered nerve, II grouptissue-engineered nerve, sciatic nerves of homogeneous SD rats weresubcutaneously embed in order. After SD rats bred7days, take out allnerves segments for microscope observation and count of CD4+and CD8+lymphocyte after HE dyeing.Results: After being chemically extracted, on intersected slices,epineurium and perineurium of acellular nerve graft formed a circularstructure, to be filled with loose collagen structure inside, having no myelinor cell components. The nerve membrane and cellular basement membranekept complete. On the longitudinal slices, many pipe-like structures neatlyarranged without cell element. After injected the neural stem cells, more cells components uneven distributed under epineurium and betweenperineurium on the longitudinal slices. after cultured1week, both groupshad more proliferation and differentiation nerve cells, NSCs geneticallymodified grew well in the acellular nerve bridge graft of adult SD rats, andmigrated in rows. But II group was more remarkable, and there wasstatistical difference between I group and II group(P<0.05). After neuralsegments subcutaneously embed in SD rats for7days, blue lymphocytesdecreased in order under microscope. And there were statistical differencesamong A、B、C groups,while having no statistical differences between Cand D group. For CD4+and CD8+lymphocytes in nerves, A group wasmost, D group was least, and there were statistical differences among A、B、C groups,while having no statistical differences between C and D group.Conclusions: Acellular nerve graft chemically extracted have no cellsand myelin components, while remaining complete neural basementmembrane and framework, moreover, the main element of basementmembrane-laminin almost keep virgin. Tissue-engineered nerve bridginggraft transplanted NSCs and cultured in medium including TMP have lowimmunogenicity and three-dimensional structure of normal nerves, maybebecome a ideal tissue-engineered artificial nerve bridging graft forrepairing peripheral nervous defection. PART3EXPERIMENTAL STUDY OF TISSUE-ENGINEERED NERVE BRIDGING GRAFT AND TMPREPAIRING ISCHIADIC NERVE DEFECTS ON RATSPurpose: Research the effect of tissue-engineered nerve bridging graftand TMP repairing large ischiadic nerve defects on rats.Methods: According to the front methods, product NSCs of geneticalmodification, Acellular sciatic nerve graft of Wistar rats, Tissue-engineered nerve bridging graft transplanted NSCs and cultured in mediumincluding TMP for1week. Divide55200-250g adult female SD rats intogroups A, B, C and D,10rats in group D and45in group A, B and Caveragely. And all experimental sciatic nerves were at the left hind side.After SD rats were anesthetized by1%pentobarbital throughabdominal injection, expose the sciatic nerves, resect the nerve1.5cmunder the piriformis lower margin for0.5cm, use4kinds of nerve grafts forbridging the sciatic nerve defects of4group SD rats: Tissue-engineerednerves transplanted NSCs and cultured in medium including TMP for1week(group A), acellular nerves group (group B), allogeneic nerves group(group C), autologous nerves group (group D). After operation, inject TMP（16mg/kg/day）in the leg triceps of A group for12weeks, only injectsame capacity0.9%NS in other3groups as well. Post-operation2weeksand12weeks, evaluate the effects of bridging grafts repairing nerve defects in every group through neurophy siological, muscle wet weight,fluorescence microscope, HRP inverse tracing and histological indicators.Results: After post-operation12weeks,in A and D groups, the digitipedis was spreading, the blottings was separating, the claws could touch theground and walk, the power of right hind limb was fine, having statisticdifference compare with B and C groups. And the sciatic nerve functionindex(SFI), the motor nerve conduction velocity and muscle wet weightrecovery rate in group A were better than group B and C (P<0.05), butslightly worse than group D (P>0.05). Through fluorescence microscopeand histological staining, NSCs in each group could survive, migrate anddifferent into neurons and glial cells in vivo, but group A was best among3groups of A、B、C. Similarly, the HRP inverse tracing indicated that theHRP positive cells in spinal ganglia of group A was more to compare withB and C groups（P<0.05）. The TB dyeing indicated that the regeneratedneurofibra and myelin of group A was more to compare with B and Cgroup while （P<0.05）.Conclusions: Tissue-engineered nerves graft transplanted NSCs andcultured in medium including TMP for1week can effectively repair sciaticnerve defects of rats and promote peripheral nerve regeneration and lowerlimb motor function recovery.